Synthetic oligonucleotides play a pivotal role in molecular biology research, useful especially for DNA sequencing, DNA amplification, and hybridization. Novel methods for the synthesis of oligonucleotides have been described previously by the inventor in International Application PCT/US93/12456 and in U.S. patent applications 08/161,224, 08/100,671, and 07/995,791 to replace both the obsolete enzymatic methods and the current chemical methods. These methods, referred to herein collectively as the "One Pot" method basically involve repeated cycles of extending an oligonucleotide primer using a nucleotide substrate having a 3'-blocking group, thus forming an extended primer with a blocking group at its 3'-end; and removal of the 3'-blocking group from the extended primer to prepare the extended primer for the addition of the next nucleotide. When the defined sequence calls for the same nucleotide to be incorporated more than once in succession, unreacted blocked nucleotide may be reused in the subsequent cycle(s). In this case, the blocking group is selectively removed from the primer-blocked nucleotide product substantially without deblocking of the unreacted blocked nucleotide. Otherwise, the method includes the further step of converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme than the blocked nucleotide. The ease with which this method can be automated will foster a new generation of oligonucleotide synthesizers with enormous throughput, increased reliability, lower cost per synthesis, and with environmentally friendly reagents.
One of the major costs associated with the enzymatic synthesis involving repetitive cycles is the cost of replenishing the enzymes for each cycle. To reduce this cost, the inventor has previously proposed that the use of a thermostable enzymes would obviate the need for replenishment after each cycle of the method.
All of the five major enzymes of the One Pot method--RNA Ligase, AMP Degrading Enzyme, Exonuclease (e.g. Phosphodiesterase I), Alkaline Phosphatase, and 3'-Phosphatase--may be used in the One Pot method as thermostable versions, provided that "significant" co-incubation of enzyme activities which could be deleterious to the synthesis is avoided. Significant co-incubation is defined by the user in terms of the desire for a certain level of product purity or product yield.
It is an object of the present invention to provide an improvement of the One Pot method in which a thermostable 3'-Phosphatase is employed.
It is a further object of the present invention to provide an improved method for synthesizing an oligonucleotide by employing a thermostable 3'-Phosphatase isolated from the hyperthermophilic archaebacterium Pyrococcus furiosus.